Interfacial activation of porcine pancreatic phospholipase A(2) studied with 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled lipids

The interfacial activation of porcine pancreatic phospholipase A(2) (PLA(2)) during the hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine liposomes at different temperatures has been monitored by fluorescence changes of the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) lipid derivatives 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (C(12)-NBD-PC) and 12-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)]dodecanoic acid (C(12)-NBD-FA) inserted in the substrate vesicles. These long-chain monitors, in contrast to the previously used C(6)-NBD-PC, detect latency times of PLA(2) action, similar to those measured by the classic titrimetric, pH-stat method. Interestingly, hydrolysis of the host vesicles results in a decrease in fluorescence not only of C(12)-NBD-PC, a substrate analog, but also of product derivative C(12)-NBD-FA. Ultrafiltration experiments show that C(12)-NBD-FA does not migrate to the aqueous phase upon hydrolysis of the host liposomes. Besides, in a simulated hydrolysis experiment in which increasing proportions of palmitic acid and 1-palmitoyl-sn-glycero-3-phosphocholine were cosonicated with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, C(12)-NBD-PC fluorescence was insensitive to products, whereas C(12)-NBD-FA did show a decreased emission intensity as in the actual hydrolysis experiments. The phenomenon is triggered above a critical concentration of products (10 mol%) suggesting that cosegregation of NBD-FA (either added as such or generated by hydrolysis of C(12)-NBD-PC) and products may be related to the decrease in fluorescence. Phase separation should create microdomains of increased C(12)-NBD-FA surface density and cause concentration quenching. In addition, and taking into account that the NBD group may be located near the interfacial region, it is possible that in segregating with products, the fluorescent moiety of C(12)-NBD-FA becomes exposed to microenvironments of higher surface polarity, which further decreases its quantum yield.